Farmed fish, such as ayu or grouper, are commercially valuable crops. A means of increasing the growth rate or size of fish crops is therefore commercially useful.
The invention is based on the discovery of three new fish growth hormone genes (from Plecoglossus altivelis, Epinephelus awoara, and Danio rerio) that can be used to increase fish crop production. For example, each of the new fish growth hormone genes can be used to generate transgenic fish expressing a fish growth hormone of the invention. These fish will exhibit greater growth rates and body sizes than the parent fish from which the transgenic fish was derived. Alternatively, the growth hormones can be recombinantly produced and introduced into the fish diet (see, e.g., Ben-Atia et al., Gen. Compar. Endocrinol. 113:155-164, 1999).
In addition, the growth hormone polypeptides and fragments thereof can be used to generate antibodies that specifically bind to a growth hormone of the invention. These antibodies can be used to, e.g., detect fish pituitary tissue in a sample. Other fragments, such as the signal sequence, can be fused to heterologous proteins to render them secretable. The growth hormone cDNA and fragments thereof can be used to screen DNA libraries (e.g., genomic or cDNA libraries) to isolate other related genes and the growth hormones which they encode.
The sequences of the new fish growth hormones are given below:
The leader peptide sequence (SEQ ID NO:3) is shown in bold, and the polyA signal is underlined. The nucleotide sequence between the initiation codon and the polyA signal, excluding the initiation codon and the polyA signal, is designated SEQ ID NO:4.
The leader peptide sequence (SEQ ID NO:7) is shown in bold, and the polyA signal is underlined. The nucleotide sequence between the initiation codon and the polyA signal, excluding the initiation codon and the polyA signal, is designated SEQ ID NO:8.
The leader peptide sequence (SEQ ID NO:11) is shown in bold, and the putative polyA signal is underlined. The nucleotide sequence between the initiation codon and the polyA signal, excluding the initiation codon and the polyA signal, is designated SEQ ID NO:12.
Accordingly, the invention features a substantially pure polypeptide having an amino acid sequence at least 75% (e.g., 80, 85, 90, 95, or 100%) identical to SEQ ID NO:2, at least 97% (e.g., 98, 99, or 100%) identical to SEQ ID NO:6, or at least 92% (e.g., 95, 98, or 100%) identical to SEQ ID NO: 10. The polypeptide increases cell proliferation, i.e., when a cell is contacted with the polypeptide in vivo or in vitro, the cell exhibits or will exhibit increased proliferation in comparison to a cell without contact with the polypeptide. Also featured is (1) a substantially pure polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of any one of SEQ ID NOs:4, 8, and 12; and (2) a substantially pure polypeptide having any one of SEQ ID NOs:2, 6, and 10 with up to 10 (e.g., 2, 4, 5, 6, or 8) conservative amino acid substitutions.
The invention includes (1) an isolated nucleic acid which hybridizes under stringent conditions to any one of SEQ ID NOs:4, 8, and 12, e.g., a nucleic acid that encodes a polypeptide that increases cell proliferation; (2) an isolated nucleic acid which hybridizes under stringent conditions to any one of SEQ ID NOs: 1, 5, and 9, where the nucleic acid is at least 20 (e.g., at least 30, 40, 50, 100, 200, and 300) nucleotides in length and does not contain 10 consecutive adenine residues; and (3) a nucleic acid encoding any polypeptide of the invention. A nucleic acid of the invention can also be less than 10,000, 1000, 500, 100, 50, or 25 thousand nucleotides in length.
In addition, the invention features a transgenic fish whose genomic DNA comprises a foreign sequence encoding a polypeptide of the invention, wherein the transgenic fish exhibits increased cell proliferation as compared to a reference fish whose genomic DNA does not have the foreign sequence. The transgenic fish can be a salmoniform (e.g., a member of the genus Plecoglossus), a perciform (e.g., a member of the genus Epinephelus), or a cypriniform (e.g., a member of the genus Danio).
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
By hybridization under xe2x80x9cstringent conditionsxe2x80x9d is meant hybridization at 65xc2x0 C., 0.5 X SSC, followed by washing at 45xc2x0 C., 0.1 X SSC.
The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264- 2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score =100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
By xe2x80x9ccell proliferationxe2x80x9d is meant an increase in cell number or cell size.
By xe2x80x9cforeign sequencexe2x80x9d as applied to a transgenic fish is meant a nucleotide sequence that does not naturally occur in the parent fish from which the transgenic fish was derived. Thus, a foreign sequence includes a non-naturally occurring additional copy of a sequence found in the parent fish or a new sequence of a fish species different from the parent fish species.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.